PROJECT SUMMARY (See Instructions); PROJECT III Implantation, a process involving the interaction between the embryonic trophectoderm and maternal endometrium, remains a fundamental barrier to successful pregnancy for many couples. However, little is known about the molecular mechanisms that regulate either tissue. MicroRNAs provide a means to understand those mechanisms. An individual microRNA can suppress hundreds of messenger RNAs, and the mlRNA-mRNA target pairs have been evolutionarily selected to have specific cellular outcomes. Therefore, uncovering miRNAs that regulate specific cell fates followed by comprehensive analysis of their downstream targets provides a window into the molecular networks that regulate those cell fates. Furthermore, understanding how miRNAs are misregulated in disease can provide insight into how dysfunction of those networks results in aberrant development. This grant proposes to use miRNAs to dissect the mechanisms regulating trophoblast stem cell proliferation and differentiation and determine whether and how these mechanisms are disrupted in pre-eclampsia, a disease associated in defects of trophoblast differentiation. The central hypothesis is that miRNA functional analyses combined with systematic target identification and functional analyses will uncover novel pathways regulating placenta growth and development. This hypothesis is supported by preliminary results showing defects in mouse trophoblast stem cells (TSC) self-renewal and differentiation associated with global miRNA loss that can be rescued by re-introduction of individual miRNAs. Three aims will be pursued. First, miRNAs that influence TSC self renewal and differentiation will be determined through rescue screens of the global knockout phenotypes, profiling, and knockdown experiments. Second, mRNA targets of miRNAs that are sufficient and/or necessary for TSC differentiation will be uncovered by genomic and high throughput functional analyses. Third, early findings will be extended to human trophoblast progenitor cells focusing on those miRNAs misregulated in patients with pre-eclampsia. The approach is innovative in its combination of miRNA functional screens, target identification, and pathway discovery in a poorly understood tissue type. It is significant as it will provide the molecular framework to understand defects in extra embryonic tissues associated with diseases of implantation and pregnancy. The proposal is part of a larger U54 center grant The proposed work will benefit greatly from this center by recruiting the other Pis' expertise in trophoblast endometrium interactions, post-transcriptional regulation, and genomic/bioinformatic approaches.